EMBO/SourceData huggingface dataset:
Figure 1(B) Representative fluorescent images (red) showing that mouse primary cortical neurons were transduced with shRNA-control or shATIC #6 at DIV 3 and then transfected with MYC-LRRK2-WT and DsRed at a plasmid ratio of 10:1 at DIV 5. Neuronal viability was analyzed at 72-hour post-transfection with non-viable neurons exhibiting no obvious neurite process (arrow). Scale bars, 100 μm. (C) Quantification of neuronal viability. Bars indicate the viability (n > 100) for each transfection condition expressed as a percent of control neurons (DsRed with pcDNA3.1 empty vector). Data represent the mean ± SEM from three independent experiments with n > 100 cells quantified in each experiment. **p < 0.01, ***p < 0.001 by one-way ANOVA followed by a Tukey's post hoc test.(D and E) Endogenous LRRK2 levels in ATIC overexpression cells. SH-SY5Y cells were transfected with V5-ATIC or empty vector. After 48 hours, LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. ***p < 0.001 by Student's t tests.(H and I) Endogenous LRRK2 levels in ATIC KO cells upon LV-flag-ATIC expressing. SH-SY5Y WT or ATIC KO cells were infected with lentiviruses carrying Flag-ATIC or empty vector. After 48 hours, LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are the means ± SEM, n=3 independent experiments. ***p < 0.001 by one-way ANOVA followed by a Tukey's post hoc test.
Figure 2(A and B) Overexpressed LRRK2 levels in HEK 293T cells upon AICAr treatment. HEK293 cells were transfected with MYC-LRRK2 and treated with or without 1mM AICAr for 48 hours. LRRK2 levels were analyzed by Western blot.(G-N) LRRK2 expression levels in primary neurons (G and H), microglia (I and J), astrocytes (K and L) and human fibroblasts (M and N) upon AICAr treatment. Mouse primary cortical neurons, microglia, astrocytes or human fibroblasts from a health control were treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot.(Q and R) LRRK2 expression levels in mouse striatum upon AICAr treatment. AICAr (72μg/day/animal) or saline (used as a control) was injected into mouse striatum via osmotic pumps. One day after injection, striatal tissues near the injection sites were collected and LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM from total six mice (three male mice and three female mice). ****p < 0.0001 by Student's t tests.(S) Representative fluorescent images (green) showing that mouse primary cortical neurons were co-transfected with MYC-LRRK2 WT or G2019S (GS), and eGFP at a plasmid ratio of 10:1 at DIV 5, and treated with or without 1mM AICAr. Neuronal viability was analyzed at 48-hour post-transfection with non-viable neurons exhibiting no obvious neurite process (arrow). Scale bars, 100 μm. (T) Quantification of neuronal viability. Bars indicate the viability (n > 100) for each transfection condition expressed as a percent of control neurons (eGFP with pcDNA3.1 empty vector). Data represent the mean ± SEM from three independent experiments with n > 100 cells quantified in each experiment. *p < 0.05, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by a Tukey's post hoc test.
Figure 3(A and B) LRRK2 protein levels upon treatment of AICAr together with a proteasome inhibitor MG132 or an autophagy inhibitor chloroquine (CQ). SH-SY5Y cells were treated with or without 1mM AICAr, 0.35μM MG132, and 5μM CQ as showed in (A) for 48 hours. LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by a Tukey's post hoc test.(C) LRRK2 and GBA mRNA levels upon AICAr treatment. SH-SY5Y cells were treated with or without 2mM AICAr for 24 hours. Total RNA was extracted. LRRK2 and GBA mRNA levels were analyzed by real-time PCR. Data are mean ± SEM, n=4 independent experiments. **p < 0.01 by Student's t tests. ns: not statistically significant. (D) LRRK2 mRNA level upon AICAr treatment in the presence of actinomycin D. SH-SY5Y cells were treated with or without 2mM AICAr for 24 hours and in the presence of 10μg/ml actinomycin D. Total RNA was extracted. LRRK2 mRNA levels were analyzed by real-time PCR. Data are mean ± SEM, n=4 independent experiments. **p < 0.01, ****p < 0.0001 by two-way ANOVA followed by Sidak's multiple comparison test.(E and F) LRRK2 expression levels from native LRRK2 cDNA and synthesized LRRK2 cDNA with synonymous codons upon AICAr treatment. HEK 293T cells were transfected with Flag-LRRK2 with native cDNA or with synthesized cDNA, and treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. ***p < 0.001 by Student's t tests.(I and J) The specific region (2221-3927bp) of the synthesized Flag-LRRK2 cDNA was replaced with the native sequence and the response of this chimeric LRRK2 cDNA expression to AICAr treatment was analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. ****p < 0.0001 by Student's t tests.
Figure 4(B LRRK2 protein levels in AUF1 knockout (KO) cells upon AICAr treatment. SH-SY5Y WT and AUF1 KO cells were treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot.(E and F) LRRK2 protein levels in AUF1 KO cells with AUF1 expressing back upon AICAr treatment. SH-SY5Y WT and KO cells were transfected AUF1-GFP or GFP and selected with 4μg/ml blasticidin. The resulting stable cell lines were treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot.(G and H) Overexpressed LRRK2 levels in AUF1 KO cells with AUF1 expressing back upon AICAr treatment. SH-SY5Y WT and AUF1 KO cells were transfected AUF1-GFP or GFP and selected with 4μg/ml blasticidin. The resulting cell lines were transfected with a construct with native LRRK2 cDNA or synthesized LRRK2 cDNA with synonymous codons and treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software.
Figure 5(A and B) AUF1 binds to LRRK2 mRNA upon AICAr treatment. SH-SY5Y WT and AUF1 KO cells were treated with or without 1mM AICAr for 24 hours. RNA and protein complexes were immunoprecipitated using AUF1 antibody. The LRRK2 mRNA levels were analyzed by semiquantitative reverse transcription (RT)-PCR. 18S ribosomal RNA (rRNA) was as a loading control. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. *p < 0.05 by Student's t tests.(D) Direct interaction of LRRK2 mRNA and four AUF1 isoforms. Biotin-labelled 62-nt LRRK2 RNA probes from native LRRK2 cDNA or synthesized LRRK2 cDNA were incubated with purified AUF1 isoforms in an RNA electrophoretic mobility shift assay (EMSA). Free probes and AUF1 bound probes were detected using a chemiluminescent RNA EMSA kit.
Figure 6(A and B) DCP1A and DCP2 expression levels upon AICAr treatment. SH-SY5Y cells were treated with or without 1mM AICAr for 48 hours. DCP1A and DCP2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. **p < 0.01 by Student's t tests.(E) LRRK2 mRNA decapping by DCP1A and DCP2. Recombinant DCP1A and DCP2 proteins were purified from HEK 293T cells and incubated with [α-32P]cap-labeled LRRK2 RNA. LRRK2 mRNA decapping was analyzed by one-dimensional thin layer chromatography. The decapping activity is demonstrated by the increased m7GDP levels.
Figure 7(A and B) LRRK2 protein levels upon the treatment of AICAr, AICAr plus an adenosine transporter (AT) inhibitor ABT702 or an adenosine kinase (ADK) inhibitors NBT-1. SH-SY5Y cells were treated with or without 1mM AICAr, AICAr plus 1μM ABT-702 or 50μM NBT-1 for 48 hours.(C and D) LRRK2 protein levels upon the treatment of AICAr or plus AMPK direct activator A769662. SH-SY5Y cells were treated with or without 1mM AICAr, 10, 20, or 50 μM A769662 for 48 hours.(E and F) LRRK2 protein levels upon the treatment of 1mM AICAr in WT and AMPK KO SH-SY5Ycells for 48 hours. LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software.
Figure 8(B) Representative confocal images (GFP) of dopamine neurons in each cluster from 9-week-old WT and LRRK2-G2019S transgenic flies with or without 1mM AICAr treatment after posteclosion. Scale bars, 25 μm.(C-E) Quantification of dopamine neurons per DA cluster in 9-week-old WT and LRRK2-G2019S transgenic flies with or without 1mM AICAr treatment after posteclosion. (F) Total numbers of dopamine neurons in four major DA clusters of 9-week-old WT and LRRK2-G2019S transgenic flies with or without AICAr treatment after posteclosion. Data are mean ± SEM, n=12 flies per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by a Tukey's post hoc test.
Figure 9(A) Representative images of DAneuron staining. WT and LRRK-KO mice were injected with 1ul LPS (5ug/animal) or phosphate buffered saline (PBS) into the substantia nigra pars compacta (SNpc) and administrated with AICAr (2.4μg/day/animal) or saline to same stereotaxic coordinates using osmotic pumps. After 21 days of injection, DA neurons were stained by anti-tyrosine hydroxylase (TH) antibody. Scale bars, 500 μm.(B) Quantification of TH positive neurons in substantia nigra pars compacta (SNpc) using an unbiased stereological method with stereo investigator software from 7-8 animals per group. (C) Quantification of Nissl-positive neurons in SNpc using an unbiased stereological method with stereo investigator software from 7-8 animals per group.(E) CD68 levels in WT and LRRK-KO mice injected with 1μl LPS (5μg/animal) or PBS into striatum followed by administration of AICAr (2.4μg/day/animal) or saline to same stereotaxic coordinates using osmotic pumps. After 21 days of injection, LRRK2 and CD68 expression in striatal tissues near the needle sites was analyzed by Western blot. (F) Quantification of images from (E) using ImageJ software, n=3 animals per group.
Figure 1Bright field (BF), red (mCherry) and green (Atoh1-nGFP) fluorescent images of organoid cultures after 10 days of expansion. Scale bars = 400µm.High power single plane confocal images of EdU labeled control and TRIM71-expressing organoids. An EdU pulse was given on day 8 and EdU incorporation (green) was analyzed 1.5 hours later. JAG1 immunostaining (magenta) marks supporting cells/prosensory cells, mCherry (red) marks transduced cells, Hoechst labels cell nuclei (blue). Scale bar= 25µm. Percentage of EdU+ mCherry+ cells per organoids in (I); (n = 5, two independent experiments). P-values were calculated using two-tailed, unpaired t test. **P < 0.01. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ±standard deviation (SD) of biological replicates.
Figure 2Bright field (BF), red (mCherry) and green (Atoh1-nGFP) fluorescent images of organoid cultures after 2 days of differentiation. Scale bar = 400µm.RT-qPCR of Atoh1 mRNA in organoids (n=3, two independent experiments). P-values were calculated using one-way ANOVA with Tukey's correction. ****P < 0.0001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.High power single plane confocal images of MYO7A immuno-stained (magenta) organoids. Atoh1-GFP (green) and MYO7A (magenta) mark nascent hair cells. MCherry (red) marks transduced cells. Scale bar = 20µm. Quantification of MYO7A+ mCherry+ cells per organoid in (E) (n=3, two independent experiments). P-values were calculated using one-way ANOVA with Tukey's correction. P>0.5 deemed not significant (n.s.). Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.RT-PCR of Myo7a mRNA expression in organoids (n=3, two independent experiments). P-values were calculated using one-way ANOVA with Tukey's correction. *P ≤ 0.05. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.
Figure 3Colony forming efficiency (n=4 in WT, n=5 in Trim71 KO, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. n.s, not significant. Organoid diameter (n=4 in WT, n=5 in Trim71 KO, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. n.s, not significant. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.High power single plane confocal images of EdU (red) and Hoechst (blue) labeled WT and TRIM71 KO organoids. A single EdU pulse was given on day 7 and EdU incorporation was analyzed 1.5 hours later. Scale bars = 25µm. Percentage of EdU+ cells in (F) (n=15 in WT, n=11 in Trim71 KO, three independent experiments). Two-tailed, unpaired t test was used to calculate P values, n.s. not significant. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.RT-qPCR of Trim71 and hair cell-specific (Atoh1, Gfi1 and Pou4f3) mRNAs in WT and Trim71 KO organoids after 2 days of differentiation (n=3, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. *P < 0.05. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.
Figure 4Cochlear epithelial cells isolated from P5 wild type mice were transduced with control (Ctrl) or TRIM71-expressing lentivirus and bulk RNA sequencing was used to analyze differential gene expression after 10 days of expansion. Volcano plot of RNA-seq data. Plotted is beta-value (x-axis) versus −log10 q-value (y-axis). Transcripts that are significantly upregulated in response to TRIM71 expression are marked in red circles, and transcripts that are significantly downregulated are marked in blue circles.Immunoblots showing LIN28B, AGO2, HMGA2, P-SMAD1/5/9 and β-actin protein levels in P5 control, TRIM71 and △NHL expressing cochlear organoids at 10 days of expansion. Note β-actin was used to normalize protein levels.
Figure 5Cochlear epithelial cells isolated from P5 wild type were transduced with control (Ctrl) or TRIM71-expressing lentivirus and expanded as organoids. MCherry (red) marks transduced cells. Hoechst staining (blue) labels cell nuclei. High power single plane confocal images of control (Ctrl) and TRIM71-expressing organoids immuno-stained for progenitor/supporting cell marker JAG1 (magenta) and progenitor marker HMGA2 (green) after 10 days of expansion. Scale bars=25µm. Quantification of JAG1+ mCherry+ HMGA2+ cells per organoid in (A) (n=3, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. ***P < 0.001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.Cochlear epithelial cells isolated from P5 wild type were transduced with control (Ctrl) or TRIM71-expressing lentivirus and expanded as organoids. MCherry (red) marks transduced cells. Hoechst staining (blue) labels cell nuclei. High power confocal images of control (Ctrl) or TRIM71-expressing organoids immuno-stained for progenitor/supporting cell marker JAG1 (magenta) and supporting cell marker S100A1 (green) after 10 days of expansion. Scale bars=25µm. Quantification of JAG1+ mCherry+ S100A1+ cells per organoid in (C) (n=3, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. **P < 0.01. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.Cochlear epithelial cells isolated from Atoh1-nGFP transgenic mice (E, F) were transduced with control (Ctrl) or TRIM71-expressing lentivirus and expanded as organoids. MCherry (red) marks transduced cells. Hoechst staining (blue) labels cell nuclei. High power single plane confocal images of control (Ctrl) or TRIM71-expressing organoids immuno-stained for transcription factor NFIB (magenta) after 2 days of differentiation. Atoh1-nGFP (green) marks nascent hair cells. White arrowheads mark Atoh1-GFP+ cells with low NFIB expression (NFIBlow). Scale bars=25µm. Quantification of NFIBlow, NFIBhigh, NFIBlowAtoh1-nGFP+, NFIBhigh Atoh1-nGFP+ cells in control (Ctrl) and TRIM71-expressing organoids (n=4, two independent experiments). Two-way ANOVA with Tukey's correction was used to calculate P values. ****P < 0.0001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.
Figure 6Bright field (BF) and red and green fluorescent images of organoid cultures at 2 days of differentiation. MCherry (red) marks transduced cells and Atoh1-nGFP (green) marks nascent hair cells. Scale bars =400µm.Percentage of mCherry+Atoh1-nGFP+ organoids in (B) (n=5 for Lin28b group, n=6 for all the other groups; two independent experiments). One-way ANOVA with Tukey's correction was used to calculate P values. *P ≤ 0.05, **P < 0.01 and ****P < 0.0001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.RT-qPCR- analysis of progenitor (Hmga2) and supporting cell-specific (Zbtb20, Nfib) gene expression in control, Lin28b, TRIM71 or Lin28b+TRIM71-expressing organoids after 10 days of expansion (n=3, two independent experiments). One-way ANOVA with Tukey's correction was used to calculate P values. *P ≤ 0.05, **P < 0.01 and ***P < 0.001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.
Figure 7RT-qPCR analysis of hair cell (Aoth1 and Pou4f3) and progenitor-specific (Trim71) gene expression in Hmga2 knockdown and control cultures. Hmga2 expression was analyzed to confirm knockdown. Plotted are fold difference between control and Hmga2 shRNA group and mean ± SD (n=3, biological replicates; two independent experiments). A two-tailed, unpaired t test was used to calculate P values. **P < 0.01, ***P < 0.001 and ****P < 0.0001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates. Low power bright field (BF) and green fluorescent images (Atoh1-nGFP) of Hmga2 shRNA knockdown and control organoid cultures after 2 days of differentiation. Scale bars=400 µm. Quantification of Atoh1-nGFP+ organoids in (E) (n=3, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. *P ≤ 0.05. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.
Figure 1Levels of differential free amino acids in MERRF patients' muscle (B) by LC-MS analysis, expressed relative to CTL. In panels the dashed line indicates the Mean CTL value set at 1. In panels B data are presented as Mean ± SEM. Muscle: MERRF (n=10), CTL (n=15)(H) Western blots of muscle lysates separated by denaturing SDS-PAGE and probed for OGDH, ALT2, α-actin, and GOT2 (top panel), ALT1 and GAPDH (middle panel), GOT1 and GAPDH (bottom panel), C (CTL), M (MERRF).
Figure 2(A-V) Age-dependent muscle protein levels of ALT2 (A), GOT2 (C), OGDH (E), GOT1 (G), ALT1 (I), AGC1 (K), SUCLA2 (M), BCAT2 (O), BCKDH (Q), PCCA (S), and MUT (U), estimated by band densitometry normalized by GAPDH, in COX10 KO expressed relative to same age CTL set at 1. Representative western blots of muscle lysates from 170-200 days old mice separated by denaturing SDS-PAGE and probed for ALT2 (B), GOT2 (D), OGDH (F), GOT1 (H), ALT1 (J), AGC1 (L), SUCLA2 (N), BCAT2 (P), BCKDHB (R), PCCA (T), and MUT (V) and GAPDH (B, D, F, H, J, L, N, P, R, T, V). : In panels data are presented as Mean ± SD. COX10 KO (n=4), CTL (n=4). Statistically significant differences between the two groups were estimated by unpaired two-tailed Student's test. *, p<0.05 **, p<0.005 COX10 KO vs CTL.(X) Muscle fractional incorporation of M+6 of glutamate, M+5 of αKG, M+4 of fumarate, M+4 of malate, M+3 of alanine and M+3 of lactate, in 200 days COX10 KO and CTL mice IP injected with [13C5, 15N]-glutamate. In panels X data are presented as Mean ± SEM. In panel X, COX10 KO (n=4), CTL (n=4). Statistically significant differences between the two groups were estimated by unpaired two-tailed Student's test. *, p<0.05 **, p<0.005 COX10 KO vs CTL.(Z) Muscle fractional incorporation of M+6 of valine, M+5 of α-KIV, M+4 of β-ΗΙΒ, M+3 of propionyl carnitine, in 200 days COX10 KO and CTL mice IP injected with [13C5, 15N]-valine. In panels Z, data are presented as Mean ± SEM. In panel Z, COX10 KO (n=3), CTL (n=3). Statistically significant differences between the two groups were estimated by unpaired two-tailed Student's test. *, p<0.05 **, p<0.005 COX10 KO vs CTL.
Figure 3(B) Age-dependent Ph-4EBP1/4E-BP1 ratio in COX10 KO expressed relative to same age CTL set at 1. (C) Representative western blot of muscle lysates from 200 days old mice separated by denaturing SDS-PAGE and probed for Ph-4E-BP1 and for 4E-BP1. In panels data are presented as Mean ± SD. COX10 KO (n=4), CTL (n=4). *, p<0.05 COX10 KO vs. same age CTL.(D) Age-dependent protein levels of Ph-4E-BP1 in COX10 KO muscle estimated by band densitometry normalized by GAPDH expressed relative to 50 days COX10 KO set at 1. (E) Western blots of muscle lysates from 50, 100, and 200 days old COX10 KO mice, separated by denaturing SDS-PAGE and probed for Ph-4E-BP1 and GAPDH. In panel D, data are presented as Mean ± SD. #, p<0.05 COX10 KO vs. COX10 KO at different ages. 50d (n=3), 100d (n=3), 200d (n=3).Age-dependent protein levels of SESTRIN 2 (F), REDD1 (H), LC3II (J) estimated by band densitometry normalized by GAPDH, in COX10 KO expressed relative to same age CTL set at 1. Representative western blot of muscle lysates from 100 and 170-200 days old mice separated by denaturing SDS-PAGE and probed for SESTRIN 2 (G), REDD1 (I), LC3II (K) In panels data are presented as Mean ± SD. COX10 KO (n=4), CTL (n=4). *, p<0.05 COX10 KO vs. same age CTL.Age-dependent protein levels of MURF1 (L), and ATROGIN1 (N) estimated by band densitometry normalized by GAPDH, in COX10 KO expressed relative to same age CTL set at 1. Representative western blot of muscle lysates from 100 and 170-200 days old mice separated by denaturing SDS-PAGE and probed for (M), ATROGIN1 (O), and GAPDH In panels data are presented as Mean ± SD. COX10 KO (n=4), CTL (n=4). *, p<0.05 COX10 KO vs. same age CTL.(P) Ph-4EBP1/4E-BP1 ratio and SESTRIN 2 protein levels normalized by α-actin, estimated by band densitometry, in MERRF muscle expressed relative to CTL muscle set at 1. (Q) Representative western blot of MERRF and CTL muscle lysates separated by denaturing SDS-PAGE and probed for Ph-4EBP1, 4EBP1, SESTRIN 2, and α-actin. In panel P, data are presented as Mean ± SD. MERRF (n=4), CTL (n=4). *, p<0.05 MERRF vs. CTL. Statistically significant differences between the two groups for all panels were estimated by unpaired two-tailed Student's test.